The development of drugs for the efficient treatment of intractable diseases, such as cancer and AIDS, is an important object to be achieved in the life science field. One potential method to achieve this object is using genetic medicines that act only on specific genes. In particular, an RNA interference (RNAi) method using a short double-stranded RNA 21 bases long (small interfering RNA: siRNA) has recently been attracting much attention as such a genetic medicine. The RNAi method was first reported by Fire et al. in 1998 (see Non-Patent Document 1). According to the report of Fire et al., when a double-stranded RNA of about 100 base pairs that is homologous to a specific region of a gene whose function is to be inhibited is introduced into cells, the double-stranded RNA is digested by the action of Dicer into fragments of about 20 to 25 base pairs, and then complexed with a plurality of proteins to form a RNA/protein complex (this complex is referred to as a RISC: a RNA-Induced Silencing Complex), which binds to a homologous site of mRNA produced from the target gene and thereby potently inhibits gene expression. However, it was reported that when a long double-stranded RNA of about 30 base pairs or longer is introduced into mammalian cells, an interferon response, which is an antiviral response, is induced, thus causing the phenomenon of apoptosis. Therefore, it was considered difficult to apply the RNAi method to mammals. Tuschl et al. thus chemically synthesized a 21-base-long double-stranded RNA that has dangling ends at both the 3′ ends, and reported that when such a double-stranded RNA is directly introduced into mammalian cells, the double-stranded RNA can potently inhibit gene expression sequence-specifically, while avoiding an interferon response (see Non-Patent Document 2). Tuschl et al. further synthesized short double-stranded RNAs consisting of a double-stranded region of 19 base pairs and dangling end(s) of various lengths at the 3′ or 5′ ends, and investigated their RNA interference effects. As a result, observations show that 21-base-long siRNA having a dangling end of 2 bases at both the 3′ ends demonstrated a very potent RNA interference effect, whereas no other type of short double-stranded RNA exhibited a remarkable RNA interference effect. Based on this report, the RNA interference method using a 21-base-long double-stranded RNA having a dangling end of 2 bases at both the 3′ ends is commonly used. The method of inhibiting the expression of a target gene using a short double-stranded RNA 21 bases long is herein referred to as the “siRNA method”, to distinguish it from the RNAi method.
Because the siRNA method uses synthetic RNA, sample preparation is comparatively easy, and handling is also easy; furthermore, very potent effects can be produced. Therefore, the siRNA method has been attracting much attention not only in the life science field, but also in the biotechnology business field.
However, this excellent siRNA method also has problem to be solved. As described above, siRNA is composed of an RNA molecule that is readily decomposed by the action of nuclease. Compared to single-stranded RNA, the double-stranded RNA region has a comparatively high resistance to nuclease contained in medium and/or a cell. However, a double-stranded RNA consisting of 19 base pairs scarcely produces the known RNA interference effects. As such, it has been reported that when introduced into cells containing a target gene sequence, although synthetic siRNA produces a potent gene expression-inhibitory effect for about 2 to about 4 days, its RNA interference effect is sharply reduced thereafter, and is almost completely lost in about seven days.
Various chemically modified siRNAs have recently been reported to provide synthetic siRNAs with enhanced cellular uptake efficiency and prolonged, highly active RNA interference effects. For example, to enhance the resistance to exonuclease digestion, siRNAs modified with an amino group, a thiol group, or an abasic site on the end of the siRNA have been synthesized. However, it has been reported that most of the terminally modified siRNAs 21 bases long have remarkably reduced RNA interference effects.
In recent years, J. Rossi et al. reported that a double-stranded RNA of 27 base pairs produces an RNA interference effect that is about 100 times greater than that of a double-stranded RNA of 21 base long (see Non-Patent Document 3). This is presumably because after an RNA of 27 base pairs is cleaved with an RNase III-like enzyme, Dicer, into a 21-base-long siRNA, the protein complex RISC recognizes the siRNA, so that the siRNA effects can be produced with a high efficiency.
As described above, because 27-base long RNA can produce excellent RNA interference effects, expectations to use this RNA as a genetic medicine are increasing. However, the technical method effective for enhancing the RNA interference effect of the 27-base-long RNA is completely unknown. Furthermore, the technical method for enhancing the RNA interference effect of a double-stranded RNA shorter or longer than 27 bases, which has an RNA interference effect, is also unclear.
Double-stranded RNAs having RNA interference effects are generally configured to have dangling ends. RNAs with no dangling ends (i.e., having blunt ends) have also been investigated for their RNA interference effects. The results, however, suggest that the RNA interference effects of double-stranded RNAs blunt-ended on the 5′ end side of the sense strand are substantially the same as, or lower than those of double-stranded RNAs having a dangling end on the 5′ end side of the sense strand (see Non-Patent Document 4).
Lipids have high cell membrane permeability, and are known to be useful to deliver drugs into cells. Linking such a lipid to a double-stranded RNA having an RNA interference effect is expected to increase the cellular uptake efficiency and thereby produce more potent RNA interference effects. However, it is known that when a lipid is simply linked to a double-stranded RNA having an RNA interference effect, the RNA interference effect is sharply reduced. In the prior art, a lipid-modified double-stranded RNA having both an excellent RNA interference effect and a useful effect based on a lipid had yet to be constructed.    Non-Patent Document 1: Fire et al., Nature, 391, 806-811 (1998)    Non-Patent Document 2: Tuschl et al., EMBO Journal, 20, 6877-6888 (2001)    Non-Patent Document 3: J. Rossi et al., Nature Biotech., 23, 222-226 (2005)    Non-Patent Document 4: J. T. Marques et al., Nature Biotech., 24, 559-565 (2005).